Proteolysis by the ubiquitin (Ub) system plays major roles in a multitude of processes. Substrates of the Ub-dependent, multifunctional N-end rule pathway include proteins with destabilizing N-terminal residues. Three of them, Asp, Glu and (oxidized) Cys, function through their conjugation to Arg (R), by R-transferase. Studies by our lab over the last ~3 years, made possible by this GM31530 grant, led to several discoveries. One of them is the role of the N-end rule pathway as a sensor of nitric oxide (NO), through the degradation of NO-modified regulatory proteins, including RGS4, RGS5 and RGS16 [1]. We also found (unpublished data) that R-transferase is a heme-binding enzyme, and that low-micromolar levels of heme inhibit R-transferase, apparently through a specific redox mechanism. New studies proposed in this renewal will build on these and other advances. The previously unsuspected, direct involvement of N-terminal arginylation and the N-end rule pathway in the processes mediated by nitric oxide and heme opened up several new vistas. The proposed biochemical and genetic studies are relevant to both basic biology and diseases of many organs, e.g., the pancreas, the brain, and the cardiovascular system. SPECIFIC AIMS: 1) Discovery of the physiological substrates of R-transferase (ATE1), with emphasis on substrates bearing N-terminal cysteine. 2) Applications of the recently constructed, cre-lox-based knock-in mouse mutants in which the ATE1 gene can be deleted in a controlled manner, in specific cell types of embryos or adult mice. 3) Applications of two recently constructed, tetO-based knock-in mutants in which either R-transferase or the UBR1 Ub ligase can be overexpressed in doxycycline-regulated settings, in specific mouse cell types. These "second-generation" tools (Aims 2 &3) will now be applied to dissect the N-end rule pathway in vivo. 4) Investigations of splicing-derived isoforms of R transferase, and determination of its crystal structure. 5) Studies of the interactions between heme and R-transferase (ATE1), the inhibition of R-transferase by heme, and the in vivo effects of heme on the N-end rule pathway, with emphasis on functional implications.